Acute toxicity of silver and carbon nanoaerosols to normal and cystic fibrosis human bronchial epithelial cells

Inhalation of engineered nanoparticles (NP) poses a still unknown risk. Individuals with chronic lung diseases are expected to be more vulnerable to adverse effects of NP than normal subjects, due to altered respiratory structures and functions. Realistic and dose-controlled aerosol exposures were performed using the deposition chamber NACIVT. Well-differentiated normal and cystic fibrosis (CF) human bronchial epithelia (HBE) with established air–liquid interface and the human bronchial epithelial cell line BEAS-2B were exposed to spark-generated silver and carbon nanoaerosols (20?nm diameter) at three different doses. Necrotic and apoptotic cell death, pro-inflammatory response, epithelial function and morphology were assessed within 24?h after aerosol exposure. NP exposure resulted in significantly higher necrosis in CF than normal HBE and BEAS-2B cells. Before and after NP treatment, CF HBE had higher caspase-3 activity and secreted more IL-6 and MCP-1 than normal HBE. Differentiated HBE had higher baseline secretion of IL-8 and less caspase-3 activity and MCP-1 secretion compared to BEAS-2B cells. These biomarkers increased moderately in response to NP exposure, except for MCP-1, which was reduced in HBE after AgNP treatment. No functional and structural alterations of the epithelia were observed in response to NP exposure. Significant differences between cell models suggest that more than one and fully differentiated HBE should be used in future toxicity studies of NP in vitro.

Our findings support epidemiologic evidence that subjects with chronic airway diseases are more vulnerable to adverse effects of particulate air pollution. Thus, this sub-population needs to be included in nano-toxicity studies.     

Leucine enhances aerosol performance of naringin dry powder and its activity on cystic fibrosis airway epithelial cells

The effect of different amino acids (AAs) on the aerosol performance of N spray-dried powders was studied. Morphology, size distribution, density, dissolution rate were evaluated and correlated to process parameters. The aerosol performance was analyzed by both Single Stage Glass Impinger and Andersen Cascade Impactor. Results indicated that powders containing 5% (w/w) of leucine, proline or histidine and dried from 3:7 ethanol/water feeds showed very satisfying aerodynamic properties with fine particle fraction>60%. Both neat N (raw and spray-dried) and N-leu1 dry-powder showing good aerodynamic properties were tested in cystic fibrosis (CF) and normal bronchial epithelial cells. Cell proliferation and expression levels of the key enzymes of the NF-κB and MAPK/ERK pathways, overactivated in CF cell lines, were evaluated. N-leu1 was able to significantly inhibit the expression levels of IKKα, IKKβ, as well as of the direct NF-κB inhibitor, IκBα. In addition N-Leu1 inhibited phosphorylation of ERK1/2 kinase and did not reduce cell proliferation as observed for the neat raw drug. Leucine co-spray-dried with the drug improved both aerodynamic properties and in vitro pharmacological activity of Naringin. The optimized N-Leu formulation as dry powder is potentially able to reduce hyperinflammatory status associated to CF.     

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1. Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2. By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3. The threshold concentration of guanylin causing a SCC increase was less than 50 nM, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4. Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5. The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6. Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.     

Pharmacotherapy of airway disease in cystic fibrosis

Current therapy for CF airway disease slows, but does not stop, the inexorable progression of airway damage. Recent research suggests that abnormal regulation of ion transport in CF airway epithelial cells leads to excessive Na⁺ absorption across a relatively Cl⁻¹-impermeable airway epithelium. This pattern of dysfunction predisposes to dehydration of airway secretions and adverse effects on mucociliary clearance and lung defense. W.E. Waltner et al, explain the rationale behind potential new approaches to therapy involving modification of ion channel function.     

Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo-.

1. P1 purinoceptor agonists like adenosine have been shown to stimulate Cl- transport in secretory epithelia. In the present study, we investigated whether P1 agonist-induced Cl- secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl- secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo-. 2. Addition of adenosine (ADO; 0.1-1 mM) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP approximately equal to ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo- (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl- secretion via a P1 purinoceptor. 3. The P1 agonists tested (at 0.01 and 0.1 mM) revealed a rank order of potency of 5'-N-ethylcarboxamine adenosine (NECA) > 2-chloro-adenosine (2-Cl-ADO) > R-phenylisopropyl adenosine (R-PIA). 4. The known potent A2 adenosine receptor (A2AR) agonist, 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA, 2 microM) but not the A1 adenosine receptor agonist, N6-phenyl adenosine (N6-phenyl ADO, 10 microM) markedly increased 125I efflux rate (baseline, 5.9 +/- 2.0% min-1, + CPCA, 10.9 +/- 0.6% min-1; P < 0.01). The stimulant effect of CPCA (10 microM) was abolished by addition of the A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (100 microM; reported K(i) = 11 +/- 3 microM). These results favour the involvement of A2AR. 5. ADO (0.1-mM) and CPCA (2 microM) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6-phenyl ADO did not affect [Ca2+]i. 6. In patch-clamp experiments, ADO (1 mM) induced an outwardly-rectified whole-cell Cl- current (baseline, 2.5 +/- 0.8 pA pF-1, + ADO, 78.4 +/- 23.8 pA pF-1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin-dependent protein kinase, CaMK [273-302] (20 microM), as compared to a control peptide, CaMK [284-302]. Addition of BAPTA (10 mM), a Ca2+ chelator, to the perfusion pipette also abolished the ADO-elicited Cl- current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered sensitivity to amiloride in cystic fibrosis. Observations using cultured sweat glands.

1. Using cultured epithelia from sweat glands, derived from both cystic fibrosis and normal subjects, the relationship between amiloride concentration and the inhibition of electrogenic sodium transport was measured, under short circuit conditions. 2. The Kd for amiloride in cultures from normal subjects was 0.64 microM (n = 6) while in cultures derived from CF patients the value was 1.07 microM (n = 4). The values were significantly different (P less than 0.02, ANOVA). 3. In cultures from normal sweat glands, bathed in solutions free of permeable anions (chloride/bicarbonate), the Kd for amiloride rose to a value greater than found in CF tissues (2.3 microM). In CF epithelia subject to the same conditions the abnormally high value increased further, so that in solutions without permeable anions normal and CF cultures behaved similarly. 4. In cultures derived from normal and CF tissues lowering the sodium concentration to 10 mM also lowered the Kd for amiloride, however the shift was greater for CF cultures. 5. Several possible explanations for the results are discussed. The most probable is that the relatively more positive apical membrane potential in CF epithelia opposes the interaction of amiloride with the sodium channel, implying that complex formation is potential sensitive.     

Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.

1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium.     

Synergism between interleukin (IL)-17 and toll-like receptor 2 and 4 signals to induce IL-8 expression in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is the most common lethal inherited disorder and is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The CF lung expresses a profound proinflammatory phenotype that appears to be related to a constitutive hypersecretion of interleukin (IL)-8 from airway epithelial cells in response to microbial infection. Since overproduction of IL-8 in CF contributes to massive bronchial infiltrates of neutrophils, identification of the pathways underlying IL-8 induction could provide novel drug targets for treatment of neutrophil-dominated inflammatory diseases such as CF. Here, we show that IL-17A synergistically increases IL-8 production induced by a toll-like receptor (TLR) 2 agonist, peptidoglycan (PGN), or TLR4 agonist, lipopolysaccharide (LPS), in a human CF bronchial epithelial cell line (CFBE41o-). A strong synergism was also observed in primary human CF bronchial epithelial cells, but not in human non-CF cell lines and primary cells. Notably, despite the induction of nuclear factor-κB and MAP kinases during TLR2 or TLR4 activation in CFBE41o-, IL-17A-dependent synergism appears to be the result of enhanced PGN- or LPS-induced phosphorylation of p38. Taken together, these studies provide evidence that IL-17A is a critical factor in increasing IL-8 expression in bacteria-infected CF airways via a pathway that regulates p38 phosphorylation.     

Guanabenz, an alpha2-selective adrenergic agonist, activates Ca2+-dependent chloride currents in cystic fibrosis human airway epithelial cells

In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca(2+)-stimulated Cl(-) secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl(-) conductance. Here, using a simple high-throughput screening assay to search for Cl(-) channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin((R))), an alpha(2)-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC(50)=831 nM) a DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) sensitive and Ca(2+) dependent Cl(-) channel (CaCC). Guanabenz activated a linear Cl(-) channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca(2+) concentration stimulating an influx of Ca(2+). In the absence of extracellular Ca(2+), the guanabenz effects on Ca(2+) influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca(2+)-dependent Cl(-) channels via a Ca(2+) influx in human cystic fibrosis airway epithelial cells.     

Immunology of cystic fibrosis.

The combination of all the immunological abnormalities described in CF appear to have a final common pathway through effects on neutrophils. Potent neutrophil chemotactic factors are produced as a result of antibody-antigen interactions leading to complement activation and the generation of C5a and the many cytokines released as a result of cellular immune responses leading to neutrophil influx and activation. In addition, the product of bacterial metabolism fMLP also produces neutrophil chemotaxis. The activated neutrophils release a range of proteases and oxygen radicals which directly damage tissues. It can, therefore, be hypothesised that the excessive immune response is directly contributing to the tissue damage. Indeed, it is even possible that this is the principal cause of the lung function defect without invoking any direct influence of the infecting organisms. This, of course, has major implications for approaches to therapy. Currently the main focus is on suppression of the organisms. However, modulation of the immune response is an attractive alternative approach.     

Taurocholic acid-induced secretion in normal and cystic fibrosis mouse ileum

Bile acids cause secretion throughout the intestinal tract and this process contributes to maintaining the fluidity of intestinal contents. In cystic fibrosis (CF) defective intestinal secretion can lead to excessive dehydration of the luminal contents and the development of clinical symptoms. This study was designed to investigate bile acid-induced secretion in mouse ileum and to determine whether this process was defective in CF. Taurocholic acid-induced secretion was monitored as a rise in short-circuit current (SCC) in ileal sheets from normal (Swiss MF1) and transgenic CF mice. Taurocholic acid increased the SCC in both intact and stripped ileal sheets from Swiss MF1 mice. This effect was due to a stimulation of electrogenic Cl- secretion as it was inhibited by Cl(-)-free conditions, serosal furosemide (frusemide), mucosal diphenylamine-2-carboxylic acid (DPC) and increased serosal K+ concentration, without being affected by reduced mucosal Na+ concentration. Taurocholic acid-induced secretion was inhibited by tetrodotoxin, indicating the involvement of a neural pathway, but this did not include capsaicin-sensitive afferent neurons or muscarinic cholinoreceptors. Mucosal mast cells also contributed to the response. Responses in tissues from transgenic wild-type mice were similar to those obtained with Swiss MF1 animals, but ilea from CF mice exhibited a lower basal SCC with significantly reduced secretory responses to acetylcholine and taurocholic acid. We concluded that taurocholic acid induces ileal secretion by a mechanism that entails activation of enteric nerves and degranulation of mucosal mast cells. Impaired bile acid-induced secretion in CF may contribute to luminal dehydration.     

Inflammatory response modulation of airway epithelial cells exposed to formaldehyde

The two main difficulties when assessing the role and action mechanism of environmental pollutant exposure on the respiratory tract using in vitro methodology are firstly to create exposure conditions that closely mimic the human situation, and secondly to choose an experimental model that accurately represents lung compartment complexity, with different types of cell interaction. The aim of this study was to resolve these two challenges. The first of our difficulties was to find the closest experimental conditions to mimic respiratory environmental pollutant exposure. We compared the effects of formaldehyde (FA) on two cellular models, alveolar and bronchial cell lines, respectively A549 and BEAS-2B. The cells were exposed for 30 min to an environmental dose of gaseous FA (50 μg/m3) at the air-liquid interface. In order to mimic macrophage-epithelial cell cooperation, sensitizations (with TNFα or with conditioned medium from macrophages--CM) prior to gas exposure were applied. After toxicity evaluation, local inflammation was assessed by IL-8 and MCP-1 production 24h after exposure. In our experimental conditions FA had no effects on alveolar and bronchial epithelial cells without any sensitization. FA exposure after TNFα sensitization alone induced a moderate increase of IL-8 by A549 cells. After sensitization with CM, FA exposure induced a strong increase of IL-8 production by A549 cells in comparison to air, whereas a decrease of MCP-1 production was observed on BEAS-2B cells. It appears that the response of alveolar and bronchial epithelial cells to FA was moderate and that complex sensitization refines the inflammatory response to environmental stresses. When sensitized with CM, these cell lines responded differently to FA exposure. Finally by interacting with the respiratory epithelium, FA could exacerbate the inflammation of airways that occurs in severe asthma, and even synergize the effects of other air pollutants such as allergens. Evaluation of nasal cell inflammatory response could shed further light on the effects of FA on respiratory epithelium.     

丙酸氟替卡松对呼吸道上皮细胞基因表达的不同作用

目的呼吸道上皮细胞在固有免疫和炎症反应中都扮演重要角色。本文旨在研究强效吸入型糖皮质激素丙酸氟替卡松(FP)对双链RNA(dsRNA,Toll样受体3的配体)刺激的呼吸道粘膜上皮细胞BEAS-2B产生的某些固有免疫相关蛋白和炎性细胞因子的作用。方法FP(10-7mol.L-1)作用于BEAS-2B 2 h后,加入dsRNA 25 mg.L-1继续培养18 h,提取细胞RNA和收集细胞培养上清液。实时定量PCR测定炎性细胞因子IL-8、GM-CSF、IL-1β和固有免疫蛋白血清淀粉样蛋白(SAA)、补体B因子、分泌性白细胞蛋白酶抑制物(SLPI)的mRNA含量;ELISA分析上清液中IL-8、GM-CSF、SAA蛋白水平。结果BEAS-2B细胞在dsRNA刺激后,IL-8、GM-CSF、IL-1β、SAA、补体B因子、SLPI的mRNA均增高。FP预处理的细胞,在dsRNA刺激后,SAA、补体B因子、SLPI的mRNA水平不降低,IL-8、GM-CSF、IL-1β的mRNA水平明显降低。IL-8、GM-CSF、SAA的ELISA分析证实了mRNA的试验结果。结论FP在强效抗炎的同时,并不损伤呼吸道上皮局部的固有免疫。     

Screening for cystic fibrosis.

Neonatal screening for cystic fibrosis (CF) reduces short-term morbidity but its long term effects remain to be demonstrated. The best available method is the assay of immunoreactive trypsin in dried blood spots, and specificity can be improved by adding direct or indirect genetic analysis. Pregnancies known to be at risk of CF can also be screened by molecular methods, and affected pregnancies terminated. The application of genetic testing to whole communities, to detect unknown heterozygotes, raises many questions which require consideration by society and the health professions. The development of effective treatment of the basic abnormality of cell function in CF would enhance the need for neonatal screening, and possibly reduce the requirement for abortion.     

Detection of sputum eicosanoids in cystic fibrosis and in normal saliva by bioassay and radioimmunoassay.

We have measured arachidonic acid (AA) metabolites, leukotrienes (LTs) and prostanoids (Ps), in sputum of patients with cystic fibrosis (CF) and in normal saliva using bioassay and radioimmunoassay (RIA). Almost three times as much LTB4 is present in CF extracts compared with slow reacting substances (SRSs). Leukotrienes were not detected in normal saliva. In CF sputum there is a three-fold increase in the level of the vasodilator prostanoid prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-oxo PGF1 alpha compared with the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TxB2), a hydrolysis product of thromboxane A2. Experiments with BW755c (25 micrograms ml-1, n = 3) indicated that the majority of this activity was not produced during the extraction procedure. The detection of LTs and Ps in sputum of CF patients shows that these substances are present at biologically active concentrations and may contribute to the pathophysiology of this disease.